Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus

Abstract

, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.

Involvement of amfC in physiological and morphological development in Streptomyces coelicolor A3(2) The GenBank accession number for the amfC promoter sequence reported in this paper is D63677.

“…However, at 9 d after inoculation, M130∆amfC began to produce undecylprodigiosin, indicating that the amfC mutation delayed but did not prevent production. All of these observations suggested that amfC mutations in strain M130 influenced secondary metabolism as well as morphogenesis, in contrast to the situation in S. griseus where amfC mutations caused no detectable effects on streptomycin or A-factor production (Kudo et al, 1995).…”

“…Disruption of the chromosomal amfC in wild-type S. griseus severely reduced the abundance of spores due to infrequent sporulation. Nucleotide sequences homologous to amfC are distributed in all 12 Streptomyces species tested (Kudo et al, 1995), which suggests a common role of amfC. An AmfC homologue (222 aa) in Streptomyces coelicolor A3(2) shows 60 % identity in amino acid sequence to AmfC in S. griseus.…”

“…S. coelicolor A3(2) strains M130 (hisA1 uraA1 SCP1 − SCP2 − ) and M145 (sre-1 SCP1 − SCP2 − ) and S. lividans strains TK21 and TK24 were obtained from D. A. Hopwood, John Innes Research Centre, Norwich, UK (Hopwood et al, 1985). A pUC19 derivative containing the whole amfC sequence in a 2n7 kb BamHI fragment (Kudo et al, 1995) was used as a source of amfC from S. coelicolor A3(2) M130. Other Streptomyces strains were obtained from IFO.…”

“…Screening a DNA library of chromosomal genes from the wild-type strain for DNA fragments that restored aerial mycelium formation in the A-factor-deficient mutant strain HH1 has led to the isolation of an operon and two genes : amfR-amfA-amfB (Ueda et al, 1993(Ueda et al, , 1998, orf1590 (Babcock & Kendrick, 1990 ;Ueda et al, 1993) and amfC (Kudo et al, 1995). amfC encodes a 218 aa protein that does not restore A-factor or streptomycin production, indicating that this gene acts in aerial mycelium formation independently of its secondary metabolic function (Kudo et al, 1995). Disruption of the chromosomal amfC in wild-type S. griseus severely reduced the abundance of spores due to infrequent sporulation.…”

A putative regulatory element for carbon-source-dependent differentiation in Streptomyces griseus b bThe GenBank accession number for the sequence reported in this paper is AB023642.

“…amfR, encoding a putative response regulator of two-component regulatory systems, is believed to act positively on the initiation of developmental processes ; its disruption abolished aerial mycelium and spore formation completely (Ueda et al, 1998). amfC cloned separately on a high-copy-number plasmid had a similar suppressive effect, and null mutation also resulted in significant loss of sporulation efficiency (Kudo et al, 1995). These results indicate that introduction of a positive regulatory gene at a high copy number may compensate for the original defects in the signal transduction system and result in the wild phenotype.…”